Antineoplastic combinations with mTOR inhibitor, trastuzumab and/or HKI-272

ABSTRACT

A combination of temsirolimus and trastuzumab in the treatment of cancer is provided. A combination of temsirolimus and HKI-272 is provided. A combination of a trastuzumab and a HKI-272 is also provided. Regimens and kits for treatment of metastatic breast cancer, containing trastuzumab, temsirolimus and/or HKI-272, optionally in combination with other anti-neoplastic agents, or immune modulators are described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/454,768, filed Apr. 24, 2012, which is a continuation of U.S. patentapplication Ser. No. 12/539,914, filed Aug. 12, 2009, which is adivision of U.S. patent application Ser. No. 11/592,066, filed Nov. 2,2006, which claims the benefit under 35 USC 119(e) of U.S. ProvisionalPatent Application No. 60/837,509, filed Aug. 14, 2006 and U.S.Provisional Patent Application No. 60/733,562, filed Nov. 4, 2005. Theforegoing related applications, in their entirety, are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

This invention relates to the use of combinations of trastuzumab with anmTOR inhibitor and/or a HKI-272, for the treatment of neoplasmsassociated with overexpression or amplification of HER2.

CCI-779, rapamycin 42-ester with3-hydroxy-2-(hydroxymethyl)-2-methylpropionic acid, is an ester ofrapamycin which has demonstrated significant inhibitory effects on tumorgrowth in both in vitro and in vivo models. This compound is now knowngenerically under the name temsirolimus. The preparation and use ofhydroxyesters of rapamycin, including temsirolimus, are described inU.S. Pat. Nos. 5,362,718 and 6,277,983.

Temsirolimus exhibits cytostatic, as opposed to cytotoxic properties,and may delay the time to progression of tumors or time to tumorrecurrence. Temsirolimus is considered to have a mechanism of actionthat is similar to that of sirolimus (rapamycin). Temsirolimus binds toand forms a complex with the cytoplasmic protein FKBP, which inhibits anenzyme, mTOR (mammalian target of rapamycin, also known asFKBP12-rapamycin associated protein [FRAP]). Inhibition of mTOR's kinaseactivity inhibits a variety of signal transduction pathways, includingcytokine-stimulated cell proliferation, translation of mRNAs for severalkey proteins that regulate the G1 phase of the cell cycle, andIL-2-induced transcription, leading to inhibition of progression of thecell cycle from G1 to S. The mechanism of action of temsirolimus thatresults in the G1-S phase block is novel for an anticancer drug.

Metastatic breast cancer (MBC) is essentially incurable with standardtherapy, and patients with MBC have a median survival of about 2 yearsafter documentation of metastasis. As a consequence, the goals oftreatment are to improve patients' symptoms while trying to maintain (orimprove, in certain cases) quality of life. Prolonging survival remainsa clear goal, particularly in breast cancer that has overexpression oramplification of the her-2 oncogene.

Herceptin® trastuzumab is an FDA-approved therapeutic monoclonalantibody for HER2 protein overexpressing metastatic breast cancer. Amurine monoclonal antibody was described [see, U.S. Pat. No. 5,705,151].The murine MAb4D5 molecule described in that document has been humanizedin an attempt to improve its clinical efficacy by reducingimmunogenicity and allowing it to support human effector functions. WO92/22653. Later documents describe the development of a lyophilizedformulation comprising full length humanized antibody huMAb4D5-8described in WO 92/22653. Herceptin® trastuzumab is currently approvedby the FDA for the treatment of metastatic breast cancer thatoverexpresses HER2—(1) as a single agent after previous treatment of themetastatic breast cancer with one or more chemotherapy regimens and (2)in combination with paclitaxel in such patients without priorchemotherapy for their metastatic breast cancer. Moreover, there isevidence that the addition of trastuzumab to taxane adjuvant orneoadjuvant chemotherapy improves to patients with earlier stage breastcancer.

HKI-272, (E)-N-{4-[3-chloro-4-(2-pyridinylmethoxy)anilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamide,has been described as a promising anticancer drug candidate for thetreatment of breast cancers and other HER-2-dependent cancers. Becauseit also inhibits the EGFR kinase with similar potency, HKI-272 may beuseful to treat tumors that overexpress both HER-2 and EGFR and be moreefficacious than a specific EGFR or HER-2 antagonist. S. K. Rabindran etal, “Antitumor Activity of HKI-272, an Orally Active, IrreversibleInhibitor of the HER-2 Tyrosine Kinase”, Cancer Research 64, 3958-3965,Jun. 1, 2004. See, U.S. Pat. No. 6,288,082; 6,297,258.

What is needed is an improved antineoplastic therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a 3-dimensional contour plot with the plane at 0% representingadditive interaction, and peaks and valleys representing areas ofsynergy or antagonism, respectively, between the Herceptin® trastuzumab(“Herceptin”) and HKI-272 in BT474 [HER-2+ (amplified); ATCC HTB-20]cells.

FIG. 2 is a 3-dimensional contour plot with the plane at 0% representingadditive interaction, and peaks and valleys representing areas ofsynergy or antagonism between the Herceptin® trastuzumab (“Herceptin”)and HKI-272 in MCF-7 [HER-2⁻, EGFR−; adenocarcinoma; ATCC HTB22] cells.

FIG. 3 is a 3-dimensional contour plot with the plane at 0% representingadditive interaction, and peaks and valleys representing areas ofsynergy or antagonism between the Herceptin® trastuzumab (“Herceptin”)and HKI-272 in MDA-MB-361 [HER-2⁺ (non-amplified); adenocarcinoma; ATCCHTB 27] cells.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides the use of combinations comprising atrastuzumab, an mTOR inhibitor and/or a HKI-272 in the treatment ofneoplasms. Thus, the invention provides for the combined use of atrastuzumab with an mTOR inhibitor, the combined use of trastuzumab witha HKI-272, the combined use of an mTOR inhibitor with a HKI-272, or thecombined use of a trastuzumab with mTOR inhibitor and a HKI-272. Theinvention further provides products containing a trastuzumab incombination with an mTOR inhibitor and/or a HKI-272 formulated forsimultaneous, separate or sequential use in treating neoplasms in amammal. The invention is also useful as an adjuvant and/or neoadjuvanttherapy of earlier stages of breast cancer. The following detaileddescription illustrates temsirolimus. However, other mTOR inhibitors maybe substituted for temsirolimus in the methods, combinations andproducts described herein.

These methods, combinations and products are useful in the treatment ofa variety of neoplasms associated with overexpression or amplificationof HER2, including, for example, lung cancers, includingbronchioalveolar carcinoma and non small cell lung cancer, breastcancers, prostate cancers, myeloma, head and neck cancer, ortransitional cell carcinoma; small cell and large cell neuroendocrinecarcinoma of the uterine cervix.

In one embodiment, the combination of temsirolimus and trastuzumab isparticularly well suited for treatment of metastatic breast cancer. Inanother embodiment, the combination of trastuzumab and an mTOR inhibitorand/or a HKI-272, are well suited for treatment of neoplasms (breast,kidney, bladder, mouth, larynx, esophagus, stomach, colon, ovary, andlung), and polycystic kidney disease.

As used herein, the term mTOR inhibitor means a compound or ligand, or apharmaceutically acceptable salt thereof, that inhibits cell replicationby blocking the progression of the cell cycle from G1 to S. The termincludes the neutral tricyclic compound rapamycin (sirolimus) and otherrapamycin compounds, including, e.g., rapamycin derivatives, rapamycinanalogues, other macrolide compounds that inhibit mTOR activity, and allcompounds included within the definition below of the term “arapamycin”. These include compounds with a structural similarity to “arapamycin”, e.g., compounds with a similar macrocyclic structure thathave been modified to enhance therapeutic benefit. FK-506 can also beused in the method of the invention.

As used herein, the term a rapamycin defines a class ofimmunosuppressive compounds that contain the basic rapamycin nucleus asshown below.

The rapamycins of this invention include compounds that are chemicallyor biologically modified as derivatives of the rapamycin nucleus, whilestill retaining immunosuppressive properties. Accordingly, the term arapamycin includes rapamycin, and esters, ethers, carbamates, oximes,hydrazones, and hydroxylamines of rapamycin, as well as rapamycins inwhich functional groups on the rapamycin nucleus have been modified, forexample through reduction or oxidation. Also included in the term arapamycin are pharmaceutically acceptable salts of rapamycins.

The term a rapamycin also includes 42- and/or 31-esters and ethers ofrapamycin as described in the following patents, which are all herebyincorporated by reference: alkyl esters (U.S. Pat. No. 4,316,885);aminoalkyl esters (U.S. Pat. No. 4,650,803); fluorinated esters (U.S.Pat. No. 5,100,883); amide esters (U.S. Pat. No. 5,118,677); carbamateesters (U.S. Pat. No. 5,118,678); silyl esters (U.S. Pat. No.5,120,842); aminodiesters (U.S. Pat. No. 5,162,333); sulfonate andsulfate esters (U.S. Pat. No. 5,177,203); esters (U.S. Pat. No.5,221,670); alkoxyesters (U.S. Pat. No. 5,233,036); O-aryl, -alkyl,-alkenyl, and -alkynyl ethers (U.S. Pat. No. 5,258,389); carbonateesters (U.S. Pat. No. 5,260,300); arylcarbonyl and alkoxycarbonylcarbamates (U.S. Pat. No. 5,262,423); carbamates (U.S. Pat. No.5,302,584); hydroxyesters (U.S. Pat. No. 5,362,718); hindered esters(U.S. Pat. No. 5,385,908); heterocyclic esters (U.S. Pat. No.5,385,909); gem-disubstituted esters (U.S. Pat. No. 5,385,910); aminoalkanoic esters (U.S. Pat. No. 5,389,639); phosphorylcarbamate esters(U.S. Pat. No. 5,391,730); carbamate esters (U.S. Pat. No. 5,411,967);carbamate esters (U.S. Pat. No. 5,434,260); amidino carbamate esters(U.S. Pat. No. 5,463,048); carbamate esters (U.S. Pat. No. 5,480,988);carbamate esters (U.S. Pat. No. 5,480,989); carbamate esters (U.S. Pat.No. 5,489,680); hindered N-oxide esters (U.S. Pat. No. 5,491,231);biotin esters (U.S. Pat. No. 5,504,091); O-alkyl ethers (U.S. Pat. No.5,665,772); and PEG esters of rapamycin (U.S. Pat. No. 5,780,462). Thepreparation of these esters and ethers is disclosed in the patentslisted above.

Further included within the definition of the term a rapamycin are27-esters and ethers of rapamycin, which are disclosed in U.S. Pat. No.5,256,790. Also described are C-27 ketone rapamycins which are reducedto the corresponding alcohol, which is in turn converted to thecorresponding ester or ether. The preparation of these esters and ethersis disclosed in the patent listed above. Also included are oximes,hydrazones, and hydroxylamines of rapamycin are disclosed in U.S. Pat.Nos. 5,373,014, 5,378,836, 5,023,264, and 5,563,145. The preparation ofthese oximes, hydrazones, and hydroxylamines is disclosed in theabove-listed patents. The preparation of 42-oxorapamycin is disclosed inU.S. Pat. No. 5,023,263.

As used herein, the term a CCI-779 means rapamycin 42-ester with3-hydroxy-2-(hydroxymethyl)-2-methylpropionic acid (temsirolimus), andencompasses prodrugs, derivatives, pharmaceutically acceptable salts, oranalogs thereof.

Examples of a rapamycin include, e.g., rapamycin, 32-deoxorapamycin,16-pent-2-ynyloxy-32-deoxorapamycin,16-pent-2-ylyloxy-32(S)-dihydro-rapamycin,16-pent-2-ylyloxy-32(S)-dihydro-40-O-(2-hydroxyethyl)-rapamycin,40-O-(2-hydroxyethyl)-rapamycin, rapamycin 42-ester with3-hydroxy-2-(hydroxymethyl)-2-methylpropionic acid (CCI-779),40-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]-rapamycin, or apharmaceutically acceptable salt thereof, as disclosed in U.S. Pat. No.5,362,718, ABT578, or 40-(tetrazolyl)-rapamycin,40-epi-(tetrazolyl)-rapamycin, e.g., as disclosed in InternationalPatent Publication No. WO 99/15530, or rapamycin analogs as disclosed inInternational Patent Publication No. WO 98/02441 and WO 01/14387, e.g.,AP23573. In another embodiment, the compound is Certican™ everolimus(42-O-(2-hydroxy)ethyl rapamycin), Novartis, U.S. Pat. No. 5,665,772.

As used herein, a HKI-272 refers to a compound having the followingcore,

or a derivative or pharmaceutically acceptable salt thereof. Suitablederivatives may include, e.g., an ester, ether, or carbamate. The corestructure, HKI-272, has the chemical name(E)-N-{4-[3-chloro-4-(2-pyridinylmethoxy)anilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamide.

In one embodiment, the invention also provides for use of substituted3-cyano quinolines having structure:

-   -   where R₁ is halogen;    -   R₂ is pyridinyl, thiophene, pyrimidine, thiazole, or phenyl        optionally substituted with up to three substituents;    -   R₃ is —O— or —S—;    -   R₄ is methyl or CH₂CH₂OCH₃;    -   R₅ is ethyl or methyl; and    -   n is 0 or 1.        These compounds, of which HKI-272 is a species, are        characterized by the ability to act as potent HER-2 inhibitors.        See, e.g., U.S. Pat. Nos. 6,288,082 and 6,297,258. These        compounds and their preparation are described in detail in US        Published Patent Application No. 2005/0059678. For convenience,        HKI-272 is used throughout this specification. However, it will        be understood that the compound of the structure provided above        can be substituted for HKI-272 in the combinations with an mTOR        inhibitor and/or a trastuzumab which are described in detail        below.

The following standard pharmacological test procedure can be used todetermine whether a compound is an mTOR inhibitor, as defined herein.Treatment of growth factor stimulated cells with an mTOR inhibitor likerapamycin completely blocks phosphorylation of serine 389 as evidencedby Western blot and as such constitutes a good assay for mTORinhibition. Thus, whole cell lysates from cells stimulated by a growthfactor (e.g., IGF1) in culture in the presence of an mTOR inhibitorshould fail to show a band on an acrylamide gel capable of being labeledwith an antibody specific for serine 389 of p70s6K.

It is preferred that the mTOR inhibitor used in the antineoplasticcombinations of this invention is a rapamycin, and more preferred thatthe mTOR inhibitor is rapamycin, temsirolimus, or everolimus. Thepreparation of everolimus is described in U.S. Pat. No. 5,665,772.

The preparation of temsirolimus is described in U.S. Pat. No. 5,362,718.A regiospecific synthesis of temsirolimus is described in U.S. Pat. No.6,277,983, which is hereby incorporated by reference. Still anotherregiospecific method for synthesis of temsirolimus is described in USPatent Publication No. 2005-0033046-A1, published Feb. 10, 2005(application Ser. No. 10/903,062, filed Jul. 30, 2004), and itscounterpart, International Patent Publication No. WO 2005/016935,published Apr. 7, 2005.

Trastuzumab, and methods of making and formulating same have beendescribed. See, e.g., U.S. Pat. Nos. 6,821,515; 6,399,063 and 6,387,371.Trastuzumab is available commercially from Genentech. As used herein,the term a trastuzumab includes trastuzumab and altered forms of, andderivatives of, trastuzumab. The term a trastuzumab includes agents thattarget the same epitope on the Her-2 receptor as targeted bytrastuzumab. The epitope is known from H. S. Cho et al., Structure ofthe extracellular region of HER2 alone and in complex with thetrastuzumab Fab, Nature 421 (2003), pp. 756-760.

HKI-272 and methods of making and formulating same have been described.See, e.g., US Published Patent Application No. 2005/0059678; U.S. Pat.No. 6,002,008, can also be used to prepare the substituted 3-quinolinecompounds used this invention and are hereby incorporated by reference.In addition to the methods described in these documents, WO-9633978 andWO-9633980 describe methods that are useful for the preparation of thesecompounds. Although these methods describe the preparation of certainquinazolines, they are also applicable to the preparation ofcorrespondingly substituted 3-cyanoquinolines and are herebyincorporated by reference.

As used in accordance with this invention, the term “treatment” meanstreating a mammal having a neoplasm by providing said mammal aneffective amount of a combination of a two or three-way combination ofthe components selected from an mTOR inhibitor, a trastuzumab and/or aHKI-272 with the purpose of inhibiting progression of the neoplasticdisease, growth of a tumor in such mammal, eradication of the neoplasticdisease, prolonging survival of the mammal and/or palliation of themammal.

As used in accordance with this invention, the term “providing,” withrespect to providing an mTOR inhibitor with a trastuzumab and/or aHKI-272, means either directly administering the mTOR inhibitor, oradministering a prodrug, derivative, or analog which will form aneffective amount of the mTOR inhibitor within the body, along with atrastuzumab and/or a HKI-272 directly, or administering a prodrug,derivative, or analog which will form an effective amount of atrastuzumab or a HKI-272 in the body.

Use of a combination of an mTOR inhibitor (e.g., temsirolimus), atrastuzumab and/or a HKI-272 also provides for the use of combinationsof each of the agents in which one, two, or all three agents is used atsubtherapeutically effective dosages. Subtherapeutically effectivedosages may be readily determined by one of skill in the art, in view ofthe teachings herein. In one embodiment, the subtherapeuticallyeffective dosage is a dosage which is effective at a lower dosage whenused in the combination regimen of the invention, as compared to thedosage that is effective when used alone. The invention further providesfor one or more of the active agents in the combination of the inventionto be used in a supratherapeutic amount, i.e., at a higher dosage in thecombination than when used alone. In this embodiment, the other activeagent(s) may be used in a therapeutic or subtherapeutic amount.

The combinations of the invention may be in the form of a kit of parts.The invention therefore includes a product containing an mTOR inhibitor,a trastuzumab and/or a HKI-272 as a combined preparation forsimultaneous, separate or sequential delivery for the treatment of aneoplasm in a mammal in need thereof. In one embodiment, a productcontains temsirolimus and a trastuzumab as a combined preparation forsimultaneous, separate or sequential use in treating a neoplasm in amammal in need thereof. Optionally, the product further contains aHKI-272. HKI-272 may be separately formulated, e.g., for oral delivery.In another embodiment, a product contains temsirolimus and a HKI-272 asa combined preparation for simultaneous, separate or sequential use in aneoplasm in a mammal in need thereof. Optionally, the product furthercontains a trastuzumab. In yet another embodiment, the product containsa trastuzumab and a HKI-272. Optionally, the product further contains anmTOR inhibitor. In one embodiment, the neoplasm is metastatic breastcancer.

In one embodiment, a pharmaceutical pack contains a course of treatmentof a neoplasm for one individual mammal, wherein the pack contains unitsof an mTOR inhibitor in unit dosage form and units of a trastuzumab inunit dosage form, optionally further in combination with units of aHKI-272 in unit dosage form. In another embodiment, a pharmaceuticalpack contains a course of treatment of a neoplasm for one individualmammal, wherein the pack contains units of an mTOR inhibitor in unitdosage form and units of a HKI-272 in unit dosage form, optionallyfurther in combination with units of a trastuzumab in unit dosage form.In yet another embodiment, a pharmaceutical pack contains a course oftreatment of a neoplasm for one individual mammal, wherein the packcontains units of a trastuzumab in unit dosage form and units of aHKI-272 in unit dosage form, optionally further in combination withunits of an mTOR inhibitor in unit dosage form. In one embodiment, apharmaceutical pack as described herein contains a course of treatmentof metastatic breast cancer for one individual mammal.

Administration of the compositions may be oral, intravenous, respiratory(e.g., nasal or intrabronchial), infusion, parenteral (besides i.v.,such as intralesional, intraperitoneal and subcutaneous injections),intraperitoneal, transdermal (including all administration across thesurface of the body and the inner linings of bodily passages includingepithelial and mucosal tissues), and vaginal (including intrauterineadministration). Other routes of administration are also feasible, suchas via liposome-mediated delivery; topical, nasal, sublingual,uretheral, intrathecal, ocular or otic delivery, implants, rectally,intranasally.

While the components of the invention may be delivered via the sameroute, a product or pack according to the invention may contain arapamycin, such as temsirolimus, for delivery by a different route thanthat of the trastuzumab or the HKI-272, e.g., one or more of thecomponents may be delivered orally, while one or more of the others areadministered intravenously. In one embodiment, temsirolimus is preparedfor oral delivery, a HKI-272 is prepared for oral delivery andtrastuzumab is prepared for intravenous delivery. In another embodiment,both temsirolimus and a trastuzumab are prepared for intravenousdelivery. In still another embodiment, all of the components areprepared for oral delivery. Optionally, other active components may bedelivered by the same or different routes as the mTOR inhibitor (e.g.,temsirolimus) or trastuzumab. Other variations would be apparent to oneskilled in the art and are contemplated within the scope of theinvention.

The mTOR inhibitor plus trastuzumab combination may be administered inthe absence of a HKI-272. In one embodiment, these are the sole activeantineoplastic agents utilized in the regimen. In another embodiment,the mTOR inhibitor/trastuzumab combination is administered incombination with HKI-272.

The mTOR inhibitor plus HKI-272 combination may be administered in theabsence of trastuzumab. In another embodiment, the mTORinhibitor/HKI-272 combination is administered in combination withtrastuzumab. In one embodiment, these two and three-way combinations arethe sole active antineoplastic agents utilized in the regimen. Inanother embodiment, these two and three-way combinations may be utilizedin further combination with other active agents.

The trastuzumab plus HKI-272 combination may be administered in theabsence of an mTOR inhibitor. In another embodiment, thetrastuzumab/HKI-272 combination is administered in combination with anmTOR inhibitor. In one embodiment, these two and three-way combinationsare the sole active antineoplastic agents utilized in the regimen. Inanother embodiment, these two and three-way combinations may be utilizedin further combination with other active agents.

As is typical with oncology treatments, dosage regimens are closelymonitored by the treating physician, based on numerous factors includingthe severity of the disease, response to the disease, any treatmentrelated toxicities, age, and health of the patient. Dosage regimens areexpected to vary according to the route of administration.

It is projected that initial i.v. infusion dosages of the mTOR inhibitor(e.g., temsirolimus) will be from about 5 to about 175 mg, or about 5 toabout 25 mg, when administered on a weekly dosage regimen. It isprojected that the oral dosage of an mTOR useful in the invention willbe 10 mg/week to 250 mg/week, about 20 mg/week to about 150 mg/week,about 25 mg/week to about 100 mg/week, or about 30 mg/week to about 75mg/week. For rapamycin, the projected oral dosage will be between 0.1mg/day to 25 mg/day. Precise dosages will be determined by theadministering physician based on experience with the individual subjectto be treated.

Other dosage regimens and variations are foreseeable, and will bedetermined through physician guidance. It is preferred that the mTORinhibitor is administered by i.v. infusion or orally, preferably in theform of tablets or capsules.

For trastuzumab, single doses and multiple doses are contemplated. Inone embodiment, a single loading dose of trastuzumab is administered asa 90-minute intravenous infusion in a range of about 4-5 mg/kg on day 1,followed by about 2 mg/kg per week starting on day 8. Typically, 3 weeksis 1 cycle. From 1, to 2 to 3, weeks may be provided between cycles.Trastuzumab may also be given at a dose of 6 mg/kg once every 3-4 weeks.In addition, trastuzumab may also be given after completion ofchemotherapy as maintenance therapy.

For a HKI-272, it is desired that a compound of the invention is in theform of a unit dose. Suitable unit dose forms include tablets, capsulesand powders in sachets or vials. Such unit dose forms may contain from0.1 to 300 mg of a compound of the invention and preferably from 2 to100 mg. Still further preferred unit dosage forms contain 5 to 50 mg ofa compound of the present invention. The compounds of the presentinvention can be administered at a dose range of about 0.01 to 100 mg/kgor preferably at a dose range of 0.1 to 10 mg/kg. In one embodiment, thecompounds are administered orally from 1 to 6 times a day, more usuallyfrom 1 to 4 times a day. Alternatively, the compounds may beadministered through another suitable route, e.g., intravenous. In stillanother embodiment, the compounds are administered once a week. Incertain situations, dosing with the HKI-272 may be delayed ordiscontinued for a brief period (e.g., 1, 2 or three weeks) during thecourse of treatment. Such a delay or discontinuation may occur once, ormore, during the course of treatment. The effective amount will be knownto one of skill in the art; it will also be dependent upon the form ofthe compound. One of skill in the art could routinely perform empiricalactivity tests to determine the bioactivity of the compound in bioassaysand thus determine what dosage to administer.

These regimens may be repeated, or alternated, as desired. Other dosageregimens and variations are foreseeable, and will be determined throughphysician guidance.

For example, in one embodiment, the regimen further comprisesadministration of a taxane, e.g., docetaxel and paclitaxel [e.g., asuspension of paclitaxel bound to albumen nanoparticles, which isavailable as Abraxane]. Paclitaxel may also be administered on a weeklyschedule, at doses 60-100 mg/m2 administered over 1 hour, weekly, or 2-3weekly doses followed by a one week rest. In one embodiment, paclitaxelis administered intravenously over 3 hours at a dose of 175 mg/m²,optionally followed by cisplatin at a dose of 75 mg/m²; or paclitaxeladministered intravenously over 24 hours at a dose of 135 mg/m²,optionally followed by cisplatin at a dose of 75 mg/m². In patientspreviously treated with therapy for carcinoma, paclitaxel can beinjected at several doses and schedules. However, the optimal regimen isnot yet clear. The recommended regimen is paclitaxel 135 mg/m² or 175mg/m² administered intravenously over 3 hours every 3 weeks. These dosesmay be altered as needed or desired.

Still other active agents may be included in a combination with an mTORinhibitor and a trastuzumab, including, e.g., chemotherapeutic agents,such as alkylating agents; hormonal agents (i.e., estramustine,tamoxifen, toremifene, anastrozole, or letrozole); antibiotics (i.e.,plicamycin, bleomycin, mitoxantrone, idarubicin, dactinomycin,mitomycin, or daunorubicin); antimitotic agents (i.e., vinblastine,vincristine, teniposide, or vinorelbine, available as Navelbine);topoisomerase inhibitors (i.e., topotecan, irinotecan, etoposide, ordoxorubicin, e.g., CAELYX or Doxil, pegylated liposomal doxorubicinhydrochloride); and other agents (i.e., hydroxyurea, altretamine,rituximab, paclitaxel, docetaxel, L-asparaginase, or gemtuzumabozogamicin); biochemical modulating agents, imatib, EGFR inhibitors suchas EKB-569 or other multi-kinase inhibitors, e.g., those that targetsserine/threonine and receptor tyrosine kinases in both the tumor celland tumor vasculature, or immunomodulators (i.e., interferons, IL-2, orBCG). Examples of suitable interferons include interferon α, interferonβ, interferon γ, and mixtures thereof.

In one embodiment, the combination of an mTOR inhibitor and atrastuzumab may be further combined with antineoplastic alkylatingagents, e.g., those described in US 2002-0198137A1. Antineoplasticalkylating agents are roughly classified, according to their structureor reactive moiety, into several categories which include nitrogenmustards, such as MUSTARGEN (meclorethamine), cyclophosphamide,ifosfamide, melphalan, and chlorambucil; azidines and epoxides, such asthiotepa, mitomycin C, dianhydrogalactitol, and dibromodulcitol; alkylsulfinates, such as busulfan; nitrosoureas, such asbischloroethylnitrosourea (BCNU), cyclohexyl-chloroethylnitrosourea(CCNU), and methylcyolohexylchloroethylnitrosourea (MeCCNU); hydrazineand triazine derivatives, such as procarbazine, dacarbazine, andtemozolomide; streptazoin, melphalan, chlorambucil, carmustine,methclorethamine, lomustine) and platinum compounds. Platinum compoundsare platinum containing agents that react preferentially at the N7position of guanine and adenine residues to form a variety ofmonofunctional and bifunctional adducts. (Johnson S W, Stevenson J P,O'Dwyer P J. Cisplatin and Its Analogues. In Cancer Principles &Practice of Oncology 6^(th) Edition. ed. DeVita V T, Hellman S,Rosenberg S A. Lippincott Williams & Wilkins. Philadelphia 2001. p.378.) These compounds include cisplatin, carboplatin, platinum IVcompounds, and multinuclear platinum complexes.

The following are representative examples of alkylating agents of thisinvention. Meclorethamine is commercially available as an injectable(MUSTARGEN). Cyclophosphamide is commercially available as an injectable(cyclophosphamide, lyophilized CYTOXAN, or NEOSAR) and in oral tablets(cyclophosphamide or CYTOXAN). Ifosfamide is commercially available asan injectable (IFEX). Melphalan is commercially available as aninjectable (ALKERAN) and in oral tablets (ALKERAN). Chlorambucil iscommercially available in oral tablets (LEUKERAN). Thiotepa iscommercially available as an injectable (thiotepa or THIOPLEX).Mitomycin is commercially available as an injectable (mitomycin orMUTAMYCIN). Busulfan is commercially available as an injectable(BUSULFEX) and in oral tablets (MYLERAN). Lomustine (CCNU) iscommercially available in oral capsules (CEENU). Carmustine (BCNU) iscommercially available as an intracranial implant (GLIADEL) and as aninjectable (BICNU). Procarbazine is commercially available in oralcapsules (MATULANE). Temozolomide is commercially available in oralcapsules (TEMODAR). Cisplatin is commercially available as an injectable(cisplatin, PLATINOL, or PLATINOL-AQ). Carboplatin is commerciallyavailable as an injectable (PARAPLATIN). Oxiplatin is commerciallyavailable as ELOXATIN.

In another embodiment, a combination of the invention may furtherinclude treatment with an antineoplastic antimetabolite, such as isdescribed in US Patent Publication No. US 2005-0187184A1 or US2002-0183239 A1. As used in accordance with this invention, the term“antimetabolite” means a substance which is structurally similar to acritical natural intermediate (metabolite) in a biochemical pathwayleading to DNA or RNA synthesis which is used by the host in thatpathway, but acts to inhibit the completion of that pathway (i.e.,synthesis of DNA or RNA). More specifically, antimetabolites typicallyfunction by (1) competing with metabolites for the catalytic orregulatory site of a key enzyme in DNA or RNA synthesis, or (2)substitute for a metabolite that is normally incorporated into DNA orRNA, and thereby producing a DNA or RNA that cannot support replication.Major categories of antimetabolites include (1) folic acid analogs,which are inhibitors of dihydrofolate reductase (DHFR); (2) purineanalogs, which mimic the natural purines (adenine or guanine) but arestructurally different so they competitively or irreversibly inhibitnuclear processing of DNA or RNA; and (3) pyrimidine analogs, whichmimic the natural pyrimidines (cytosine, thymidine, and uracil), but arestructurally different so thy competitively or irreversibly inhibitnuclear processing of DNA or RNA.

The following are representative examples of antimetabolites of thisinvention. 5-Fluorouracil (5-FU; 5-fluoro-2,4(1H,3H)-pyrimidinedione) iscommercially available in a topical cream (FLUOROPLEX or EFUDEX), atopical solution (FLUOROPLEX or EFUDEX), and as an injectable containing50 mg/mL 5-fluorouracil (ADRUCIL or fluorouracil). Floxuradine(2′-deoxy-5-fluorouridine) is commercially available as an injectablecontaining 500 mg/vial of floxuradine (FUDR or floxuradine). Thioguanine(2-amino-1,7-dihydro-6-H-purine-6-thione) is commercially available in40 mg oral tablets (thioguanine). Cytarabine(4-amino-1-(beta)-D-arabinofuranosyl-2(1H)-pyrimidinone) is commerciallyavailable as a liposomal injectable containing 10 mg/mL cytarabine(DEPOCYT) or as a liquid injectable containing between 1 mg-1 g/vial or20 mg/mL (cytarabine or CYTOSAR-U). Fludarabine(9-H-Purin-6-amine,2-fluoro-9-(5-O-phosphono-(beta)-D-a-rabinofuranosyl)is commercially available as a liquid injectable containing 50 mg/vial(FLUDARA). 6-Mercaptopurine (1,7-dihydro-6H-purine-6-thione) iscommercially available in 50 mg oral tablets (PURINETHOL). Methotrexate(MTX;N-[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-glutamicacid) is commercially available as a liquid injectable containingbetween 2.5-25 mg/mL and 20 mg-1 g/vial (methotrexate sodium or FOLEX)and in 2.5 mg oral tablets (methotrexate sodium). Gemcitabine(2′-deoxy-2′,2′-difluorocytidine monohydrochloride ((beta)-isomer)), iscommercially available as a liquid injectable containing between 200mg-1 g/vial (GEMZAR). Capecitabine(5′-deoxy-5-fluoro-N-[(pentyloxy)carbonyl]-cytidine) is commerciallyavailable as a 150 or 500 mg oral tablet (XELODA). Pentostatin((R)-3-(2-deoxy-(beta)-D-erythro-pentofuranosyl)-3,6,7,-8-tetrahydroimidazo[4,5-d][1,3]diazepin-8-ol)is commercially available as a liquid injectable containing 10 mg/vial(NIPENT). Trimetrexate(2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)methyl]quinazolinemono-D-glucuronate) is commercially available as a liquid injectablecontaining between 25-200 mg/vial (NEUTREXIN). Cladribine(2-chloro-6-amino-9-(2-deoxy-(beta)-D-erythropento-furanosyl) purine) iscommercially available as a liquid injectable containing 1 mg/mL(LEUSTATIN).

The term “biochemical modulating agent” is well known and understood tothose skilled in the art as an agent given as an adjunct to anti-cancertherapy, which serves to potentate its antineoplastic activity, as wellas counteract the side effects of the active agent, e.g., anantimetabolite. Leucovorin and levofolinate are typically used asbiochemical modulating agents for methotrexate and 5-FU therapy.Leucovorin (5-formyl-5,6,7,8-tetrahydrofolic acid) is commerciallyavailable as an injectable liquid containing between 5-10 mg/mL or50-350 mg/vial (leucovorin calcium or WELLCOVORIN) and as 5-25 mg oraltablets (leucovorin calcium). Levofolinate (pharmacologically activeisomer of 5-formyltetrahydrofolic acid) is commercially available as aninjectable containing 25-75 mg levofolinate (ISOVORIN) or as 2.5-7.5 mgoral tablets (ISOVORIN).

In another embodiment, the combination of the invention further includesan active agent selected from among a kinase inhibitor. Particularlydesirable are multi-kinase inhibitors target serine/threonine andreceptor tyrosine kinases in both the tumor cell and tumor vasculature.Examples of suitable kinase inhibitors are Sorafenib (BAY 43-9006,Bayer, commercially available as NEXAVAR), which has been granted FastTrack status by the FDA for metastatic renal cell cancer. Anothersuitable farnesyltransferase inhibitor is Zarnestra (R115777,tipifarnib). Yet another compound is suntinib (SUTENT). Still othersuitable compounds that target Ras/Raf/MEK and/or MAP kinases include,e.g., avastin, ISIS 5132, and MEK inhibitors such as CI-1040 or PD0325901.

As described herein, subtherapeutically effective amounts of trastuzumaband temsirolimus may be used to achieve a therapeutic effect whenadministered in combination. For example, trastuzumab may be provided atdosages of 5 to 50% lower, 10 to 25% lower, or 15 to 20% lower, whenprovided along with temsirolimus. For example, a resulting trastuzumabdosage can be from about 8 to 40 mg, or about 8 to 30 mg, or 8 to 25 mg.Subtherapeutically effective amounts of trastuzumab are expected toreduce the side-effects of trastuzumab treatment. The invention furtherprovides for one or more of the active agents in the combination of theinvention to be used in a supratherapeutic amount, i.e., at a higherdosage in the combination than when used alone. In this embodiment, theother active agent(s) may be used in a therapeutic or subtherapeuticamount.

The mTOR inhibitor, trastuzumab, HKI-272 or other active compounds usedin the combination and products of the invention may be formulated inany suitable manner. For example, oral formulations containing the mTORinhibitor (and optionally, other active compounds) useful in combinationand products of this invention may comprise any conventionally used oralforms, including tablets, capsules, buccal forms, troches, lozenges andoral liquids, suspensions or solutions. Capsules may contain mixtures ofthe active compound(s) with inert fillers and/or diluents such as thepharmaceutically acceptable starches (e.g. corn, potato or tapiocastarch), sugars, artificial sweetening agents, powdered celluloses, suchas crystalline and microcrystalline celluloses, flours, gelatins, gums,etc. Useful tablet formulations may be made by conventional compression,wet granulation or dry granulation methods and utilize pharmaceuticallyacceptable diluents, binding agents, lubricants, disintegrants, surfacemodifying agents (including surfactants), suspending or stabilizingagents, including, but not limited to, magnesium stearate, stearic acid,talc, sodium lauryl sulfate, microcrystalline cellulose,carboxymethylcellulose calcium, polyvinylpyrrolidone, gelatin, alginicacid, acacia gum, xanthan gum, sodium citrate, complex silicates,calcium carbonate, glycine, dextrin, sucrose, sorbitol, dicalciumphosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride,talc, dry starches and powdered sugar. Preferred surface modifyingagents include nonionic and anionic surface modifying agents.Representative examples of surface modifying agents include, but are notlimited to, poloxamer 188, benzalkonium chloride, calcium stearate,cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters,colloidal silicon dioxide, phosphates, sodium dodecylsulfate, magnesiumaluminum silicate, and triethanolamine. Oral formulations herein mayutilize standard delay or time release formulations to alter theabsorption of the active compound(s). The oral formulation may alsoconsist of administering the active ingredient in water or a fruitjuice, containing appropriate solubilizers or emulsifiers as needed.Preferred oral formulations for rapamycin 42-ester with3-hydroxy-2-(hydroxymethyl)-2-methylpropionic acid are described in USPatent Publication No. 2004/0077677 A1, published Apr. 22, 2004.

In some cases it may be desirable to administer the compounds directlyto the airways in the form of an aerosol.

The compounds may also be administered parenterally orintraperitoneally. Solutions or suspensions of these active compounds asa free base or pharmacologically acceptable salt can be prepared inwater suitably mixed with a surfactant such as hydroxy-propylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols and mixtures thereof in oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol and liquidpolyethylene glycol), suitable mixtures thereof, and vegetable oils.Preferred injectable formulations for rapamycin 42-ester with3-hydroxy-2-(hydroxymethyl)-2-methylpropionic acid are described in USPatent Publication No. 2004/0167152 A1, published Aug. 26, 2004.

For the purposes of this disclosure, transdermal administrations areunderstood to include all administrations across the surface of the bodyand the inner linings of bodily passages including epithelial andmucosal tissues. Such administrations may be carried out using thepresent compounds, or pharmaceutically acceptable salts thereof, inlotions, creams, foams, patches, suspensions, solutions, andsuppositories (rectal and vaginal).

Transdermal administration may be accomplished through the use of atransdermal patch containing the active compound and a carrier that isinert to the active compound, is non toxic to the skin, and allowsdelivery of the agent for systemic absorption into the blood stream viathe skin. The carrier may take any number of forms such as creams andointments, pastes, gels, and occlusive devices. The creams and ointmentsmay be viscous liquid or semisolid emulsions of either the oil-in-wateror water-in-oil type. Pastes comprised of absorptive powders dispersedin petroleum or hydrophilic petroleum containing the active ingredientmay also be suitable. A variety of occlusive devices may be used torelease the active ingredient into the blood stream such as asemi-permeable membrane covering a reservoir containing the activeingredient with or without a carrier, or a matrix containing the activeingredient. Other occlusive devices are known in the literature.

Suppository formulations may be made from traditional materials,including cocoa butter, with or without the addition of waxes to alterthe suppository's melting point, and glycerin. Water soluble suppositorybases, such as polyethylene glycols of various molecular weights, mayalso be used.

As used in this invention, the combination regimen can be givensimultaneously or can be given in a staggered regimen, with the mTORinhibitor being given at a different time during the course ofchemotherapy than the trastuzumab. This time differential may range fromseveral minutes, hours, days, weeks, or longer between administration ofthe at least two agents. Therefore, the term combination (or combined)does not necessarily mean administered at the same time or as a unitarydose, but that each of the components are administered during a desiredtreatment period. The agents may also be administered by differentroutes.

Pharmaceutical Packs/Kits:

The invention includes a product or pharmaceutical pack containing acourse of an anti-neoplastic treatment for one individual mammalcomprising one or more container(s) having one, one to four, or moreunit(s) of an mTOR inhibitor (e.g., temsirolimus) in unit dosage formand, optionally, one, one to four, or more unit(s) of a trastuzumab, andoptionally, another active agent.

In another embodiment, pharmaceutical packs contain a course ofanti-neoplastic treatment for one individual mammal comprising acontainer having a unit of a rapamycin in unit dosage form, a containinghaving a unit of trastuzumab, and optionally, a container with anotheractive agent. In other embodiments, the rapamycin is rapamycin, an ester(including a 42-ester, ether (including a 42-ether), oxime, hydrazone,or hydroxylamine of rapamycin. In another embodiment, the rapamycin is42-O-(2-hydroxy)ethyl rapamycin.

In another embodiment, the rapamycin is temsirolimus, and the packcontains one or more container(s) comprising one, one to four, or moreunit(s) of temsirolimus with the components described herein.

In some embodiments, the compositions of the invention are in packs in aform ready for administration. In other embodiments, the compositions ofthe invention are in concentrated form in packs, optionally with thediluent required to make a final solution for administration. In stillother embodiments, the product contains a compound useful in theinvention in solid form and, optionally, a separate container with asuitable solvent or carrier for the compound useful in the invention.

In still other embodiments, the above packs/kits include othercomponents, e.g., instructions for dilution, mixing and/oradministration of the product, other containers, syringes, needles, etc.Other such pack/kit components will be readily apparent to one of skillin the art.

The following examples illustrate of the uses of the combinations of theinvention. It will be readily understood that alterations ormodifications, e.g., in the formulation of the components, the routes ofdelivery, and the dosing, can be made for reasons known to those ofskill in the art.

Example 1: Combination Regimen of Temsirolimus (CCI-779) and Trastuzumabin Treatment of Neoplasms

Dosing begins at month 1, day 1 with weekly intravenous (IV)temsirolimus and trastuzumab (IV) at the dosages provided below.

Temsirolimus and trastuzumab can be administered simultaneously,consecutively, or on alternative days.

Temsirolimus is administered IV weekly over a 30-minute period using anin-line filter and an automatic dispensing pump. Optionally,antihistamine (diphenhydramine, 25 to 50 mg IV or the equivalent) isadministered about 30 minutes prior to temsirolimus infusion.

A trastuzumab loading dose is administered IV weekly over a 90 minuteperiod. Weekly doses are administered, which are typically half theamount of the loading dose. For example, a 4 mg/kg loading dose istypically followed by 2 mg/kg weekly doses. These amounts may beadjusted. In one embodiment, no loading dose is required and the samedose is administered throughout the course of treatment.

Trastuzumab Temsirolimus Dose (mg/kg) (mg) 2 15 4 25 6 50

Dose adjustments and/or delays for temsirolimus, and/or trastuzumab arepermitted. For example, treatment may continue as described herein forsix months, with weekly doses of temsirolimus. The trastuzumab may beprovided on a weekly basis for a cycle, e.g., three-weeks. Typically, 2to 3 weeks is provided between cycles. In certain situations, dosingwith the temsirolimus may be delayed or discontinued for a brief period(e.g., 1, 2 or three weeks) during the regimen. Similarly, a cycle oftreatment with trastuzumab may be shortened by one or more weeks,lengthened by one or more weeks, or the period between cycles delayed oreliminated. Such a delay or discontinuation may occur once, or more,during the course of treatment.

Example 2: Use of a Combination Regimen of HKI-272 and Temsirolimus(CCI-779) in Treatment of Neoplasms

Dosing begins at month 1, day 1 with daily HKI-272 and weeklyintravenous (IV) temsirolimus at the dosages provided below.

On month 1, day 1, HKI-272 is administered orally prior to temsirolimus.Temsirolimus is administered following HKI-272, preferably within 30minutes.

Temsirolimus is administered IV weekly over a 30-minute period using anin-line filter and an automatic dispensing pump. Optionally,antihistamine (diphenhydramine, 25 to 50 mg IV or the equivalent) isadministered about 30 minutes prior to temsirolimus infusion.

Thereafter, HKI-272 is taken orally once daily with food, preferably inthe morning.

HKI-272 Temsirolimus Dose (mg) (mg) 80 15 160 25 240 50

Dose adjustments and/or delays for HKI-272 and temsirolimus arepermitted. For example, treatment may continue as described herein forsix months, with daily doses of HKI-272 and weekly doses oftemsirolimus. However, in certain situations, dosing with one or bothdrugs may be delayed or discontinued for a brief period (e.g., 1, 2 orthree weeks) during the regimen course of treatment. Such a delay ordiscontinuation may occur once, or more, during the course of treatment.

Example 3: Use of a Combination Regimen of HKI-272, Temsirolimus(CCI-779), and Trastuzumab in Treatment of Neoplasms

Dosing begins at month 1, day 1 with daily HKI-272 and weeklyintravenous (IV) temsirolimus and trastuzumab (IV) at the dosagesprovided below.

On month 1, day 1, HKI-272 is administered orally prior to temsirolimus.Temsirolimus and trastuzumab are administered following HKI-272,preferably within 30 minutes.

Temsirolimus is administered IV weekly over a 30-minute period using anin-line filter and an automatic dispensing pump. Optionally,antihistamine (diphenhydramine, 25 to 50 mg IV or the equivalent) isadministered about 30 minutes prior to temsirolimus infusion.

A trastuzumab loading dose is administered IV weekly over a 90 minuteperiod. Weekly doses are administered, which are typically half theamount of the loading dose. For example, a 4 mg/kg loading dose istypically followed by 2 mg/kg weekly doses. These amounts may beadjusted. In one embodiment, no loading dose is required and the samedose is administered throughout the course of treatment.

Thereafter, HKI-272 is taken orally once daily with food, preferably inthe morning.

HKI-272 Trastuzumab Temsirolimus Dose (mg) (mg/kg) (mg) 80 2 15 160 4 25240 6 50

Dose adjustments and/or delays for HKI-272, temsirolimus, and/ortrastuzumab are permitted. For example, treatment may continue asdescribed herein for six months, with daily doses of HKI-272 and aweekly dose of temsirolimus. The trastuzumab may be provided on a weeklybasis for a cycle, e.g., three weeks. Typically, 2 to 3 weeks isprovided between cycles. However, in certain situations, dosing with theHKI-272 and/or temsirolimus may be delayed or discontinued for a briefperiod (e.g., 1, 2 or three weeks) during the regimen or course oftreatment. Such a delay or discontinuation may occur once, or more,during the course of treatment.

Similarly, a cycle of treatment with trastuzumab may be shortened by oneor more weeks, lengthened by one or more weeks, or the period betweencycles delayed or eliminated. Such a delay or discontinuation may occuronce, or more, during the course of treatment.

Example 4: Use of a Combination Regimen of HKI-272 and Trastuzumab inTreatment of Neoplasms

The antineoplastic activity of the HKI-272 plus trastuzumab combinationwas confirmed in in vitro standard pharmacological test procedure. Thefollowing briefly describes the procedure used and the results obtained.

The combination was tested in three breast cancer cell lines ofdiffering genotypes. More particularly, BT474 [HER-2+(amplified); ATCCHTB-20] and is highly sensitive to both HKI-272 and trastuzumab.MDA-MB-361 [HER-2⁺ (non-amplified); adenocarcinoma; ATCC HTB 27] haslower levels of HER-2 without amplification and less sensitive to bothtrastuzumab and HKI-272. MCF-7 [HER-2⁻, EGFR−; adenocarcinoma; ATCCHTB22] has no HER-2 and is resistant to both trastuzumab and HKI-272.

Cells from each of these cell lines were incubated in the presence of arange of concentrations (0.0041, 0.012, 0.037, 0.11, 0.33, 0.1, 3 μg/mg)for each drug. The cells were maintained in RPMI 1640 medium (LifeTechnologies, Inc., Gaithersburg, Md.) supplemented with 10% fetalbovine serum (FBS, Life Technologies) and 50 μg/ml gentamicin (LifeTechnologies) under 7% CO₂ at 37° C. Cells were plated in 96-wellmicrotiter dishes (12,000 cells/well for BT474 Cells, 6000 cells/wellMCF-7 Cells and 10,000 cells/well for MDA-MB-361 Cells) in 100 μl RPMI1640 medium containing 5% FBS and 50 g/ml gentamicin and incubatedovernight at 37° C. Compound dilutions were prepared in the same medium,at 2× final concentration, and 100 μl of the drug dilution was added tothe cell-containing wells.

Serial dilutions of one compound were prepared in the presence of afixed dose of a second compound. Alternatively, a checkerboard dilutionseries was employed. Cells were cultured for three days in the presenceof the drugs. Untreated cells were included as controls. The percentageof surviving cells was determined using sulforhodamine B (SRB,Sigma-Aldrich, St Louis, Mo.), a protein binding dye. Cellular proteinwas precipitated in each well by the addition of 50 μl 50% coldtrichloroacetic acid. After 1 hour, the plates were washed extensivelyin water and dried. SRB dye reagent (0.4% SRB in 1% acetic acid, 80 μlper well) was added and plates were kept at room temperature for tenminutes. Plates were then washed thoroughly in 1% acetic acid and dried.Cell-associated dye was dissolved in 10 mM Tris (150 μl) and theabsorbance was read at 540 nm in a microtiter plate reader. Theconcentration of compound that caused a fixed percentage inhibition ofgrowth was determined by plotting cell survival (relative to untreatedcells) against the compound dose.

A model for studying drug interactions has been described by Prichardand Shipman [Antiviral Research. 14:181-206 (1990); Prichard, Minn., etal., 1993. MacSynergy II. Version 1.0. User's manual. University ofMichigan, Ann Arbor.] This is a 3-dimensional model: one for each drugand the third for the biological effect. Theoretical additiveinteractions are calculated from the individual dose-response curves,based on a dissimilar sites model of additivity (Bliss independence).The calculated additive surface, representing predicted cytotoxicity issubtracted from the experimental surface to reveal areas of enhancedtoxicity (synergy) or reduced toxicity (antagonism). The resultingsurface appears as a horizontal plane at 0% inhibition above thecalculated additive surface, if the interaction is additive. Peaks andvalleys deviating from this plane are indicative of synergy andantagonism, respectively. MacSynergy II, a Microsoft Excel-basedsoftware was used to perform all calculations automatically. Thisspreadsheet calculates the theoretical additive interactions, andlocates and quantifies synergistic or antagonistic interactions that aresignificant at the 95% confidence levels. The results were plotted as a3-dimensional plot, or as a contour plot with the plane at 0%representing additive interaction, and peaks and valleys representingareas of synergy or antagonism, respectively, between the two drugs.

For purposes of this study, the Pritchard and Shipman method wasmodified to allow determination of the combination effects at differentlevels of statistical significance (p-values 0.05, 0.01, 0.001). Ap-value of 0.05 is considered significant. The method of estimatingstatistical variability within each experiment was also modified.Variability was determined across all compound combinations, whereas inthe original version, variability was estimated separately for eachcompound combination. It is believed that better estimates of thevariability are obtained with the modified approach. In general, singlepoints of synergy or antagonism are not considered representative ofeither synergistic or antagonistic activity. Thus, single point peaks orvalleys are disregarded in the analysis. Furthermore, peaks or valleysthat occur only along single concentration of one of the compounds arealso disregarded, if no synergy or antagonism is observed at theadjacent, flanking concentrations. Finally, all experiments are repeatedat least twice and determinations of synergy and antagonism are made byexamination of all the data.

FIGS. 1-3 provide the results from a single set of experiments. InMDA-MB-361 cells, there is an area of antagonism at 0.11-3 μg/mLtrastuzumab for at a concentration of 0.012 μg/mL HK-272 at the 95%confidence level. In MCF7 cells, there is an area of synergy at0.037-0.33 g/mL trastuzumab for at a concentration of 0.11 μg/mL HKI-272at the 95% confidence level. For the BT474 cells, there is an area ofantagonism at 0.33-1 μg/mL trastuzumab for at a concentration of 0.11μg/mL HKI-272 at the 95% confidence level. When repeated at the 99%confidence level, no statistically significant areas of antagonism orsynergy were found. Based on the above criteria, the combination oftrastuzumab and HKI-272 is considered additive across allconcentrations.

The results of these standard pharmacological test procedures derivedfrom multiple independent experiments, indicate that combinations ofHKI-272 are not significantly antagonistic or synergistic, but areadditive over a range of concentrations. These data support the use ofthe combinations in the treatment of HER2+ cancers. As thesecombinations contain at least two active antineoplastic agents, the useof such combinations also provides for the use of combinations of eachof the agents in which one or both of the agents is used atsubtherapeutically effective dosages, thereby lessening toxicityassociated with the individual chemotherapeutic agent.

All patents, patent publications, articles, and other documentsreferenced herein are incorporated by reference. It will be clear to oneof skill in the art that modifications can be made to the specificembodiments described herein without departing from the scope of theinvention.

The invention claimed is:
 1. A method of treating a HER2+ neoplasm in apatient in need thereof, consisting of administering to the patient: i)temsirolimus (CCI-779) or a prodrug or pharmaceutically acceptable saltthereof in combination with one or more pharmaceutically acceptablecarriers; and ii) 0.1 to 300 mg of(E)-N-{4-[3-chloro-4-(2-pyridinylmethoxy)anilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamide,or a pharmaceutically acceptable salt thereof, in combination with oneor more pharmaceutically acceptable carriers.
 2. The method of claim 1,wherein the temsirolimus (CCI-779) and(E)-N-{4-[3-chloro-4-(2-pyridinylmethoxy)anilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamide,or pharmaceutically acceptable salt thereof, are administered insubtherapeutically effective amounts.
 3. The method of claim 1, whereinthe neoplasm is selected from the group consisting of: lung cancer,including bronchioalveolar carcinoma and non small cell lung cancer;breast cancer; myeloma; prostate cancer; head and neck cancer;transitional cell carcinoma; and small cell and large cellneuroendocrine carcinoma of the uterine cervix.
 4. The method of claim3, wherein the breast cancer is metastatic breast cancer.
 5. The methodof claim 1, wherein the temsirolimus (CCI-779) and(E)-N-{4-[3-chloro-4-(2-pyridinylmethoxy)anilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamide,or pharmaceutically acceptable salt thereof, are administeredsimultaneously, separately, sequentially, or in a staggered regimen tothe patient.
 6. The method of claim 1, wherein the temsirolimus(CCI-779) and(E)-N-{4-[3-chloro-4-(2-pyridinylmethoxy)anilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamide,or pharmaceutically acceptable salt thereof, are administered to thepatient in a formulation suitable for one or more of the followingadministration routes: oral, as an aerosol, parenteral, intraperitoneal,transdermal, or as a suppository.
 7. The method of claim 1, wherein thetemsirolimus (CCI-779) and(E)-N-{4-[3-chloro-4-(2-pyridinylmethoxy)anilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamide,or pharmaceutically acceptable salt thereof, are administered to thepatient using the same route of administration or using different routesof administration.
 8. The method of claim 1, wherein the temsirolimus(CCI-779) and(E)-N-{4-[3-chloro-4-(2-pyridinylmethoxy)anilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamide,or pharmaceutically acceptable salt thereof, are contained in apharmaceutical pack or kit.
 9. The method of claim 1, wherein thetemsirolimus (CCI-779) is administered orally or intravenously.
 10. Themethod of claim 9, wherein an amount in the range of between 10 mg to250 mg of the temsirolimus (CCI-779) is administered orally to thepatient per week.
 11. The method of claim 9, wherein an amount in therange of between 5 mg to 175 mg of the temsirolimus (CCI-779) isadministered intravenously to the patient per week.
 12. The method ofclaim 11, wherein the(E)-N-{4-[3-chloro-4-(2-pyridinylmethoxy)anilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamide,or pharmaceutically acceptable salt thereof, is administered orally. 13.The method of claim 12, wherein the(E)-N-{4-[3-chloro-4-(2-pyridinylmethoxy)anilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamide,or pharmaceutically acceptable salt thereof, is administered daily. 14.The method of claim 13, wherein an amount of 80 mg, 160 mg, or 240 mg ofthe(E)-N-{4-[3-chloro-4-(2-pyridinylmethoxy)anilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamide,or pharmaceutically acceptable salt thereof, is administered orally.